Synthesis and properties of terthiophene-modified oligodeoxynucleotides

Synthesis and properties of oligodeoxynucleotides (ODNs) containing terthiophene (Thp) were described. One-electron oxidation of Thp-modified ODN resulted in the formation of Thp radical cation (Thp(.+)), which remained stable in the experimental time window up to 200mus, showing that charge may be carried along DNA by Thp as Thp(.+).     

Synthesis and proton-NMR studies of oligonucleotides containing an apurinic (AP) site

In order to elucidate the conformational properties of base-deleted oligodeoxyribonucleotides, the molecules d-CpS(pCpG)n (n = 1,2; S = sugar) were synthesized by the phosphotriester method and characterized by 1H-NMR spectroscopy. Complete assignment of all non-exchangeable proton resonances of both compounds was obtained by 1D- and 2D-NMR techniques. In combination with computer simulation, these spectra yielded proton-proton and proton-phosphorus coupling constants of high accuracy. These data provide valuable information about the sugar and the backbone conformation. It appears that d-Cp1Sp2Cp3G4 does not form a duplex under any of the conditions studied. On the contrary, the base-deleted hexamer d-Cp1Sp2Cp3Gp4Cp5G6 occurs as a right-handed' staggered' DNA duplex at 280 K: the core of this duplex is formed by the residues C(3)-G(6); two 'dangling' residues C(1) and S(2) are located at the two 5'-ends of the duplex. The assignment of the corresponding imino proton resonances for [d-CpS(pCpG)2]2 was based on their thermal behavior: the line broadening of these resonances was studied as a function of temperature. The chemical shift and the number of imino proton resonances accord well with the number and type of Watson-Crick base pairs which can be formed in the staggered duplex described above. Thermodynamic parameters of duplex formation were obtained from an analysis of the chemical shift versus temperature profiles of aromatic base and H-1' protons. It is suggested that the cytosine ring of C(1) stacks, at least part of the time, with the guanine ring on the nucleotide residue, G(6), situated in the complementary strand. The binding of Lys-Trp-Lys to [d-CpS(pCpG)2]2 as well as to [d-CpGpCpG]1 was investigated. It is concluded that the indole ring of the tryptophan residue probably stacks on top of the 3'-terminal guanine base of both duplexes, but not on the nucleic acid bases next to the apurinic (AP) site.     

Synthesis and binding properties of new selective ligands for the nucleobase opposite the AP site

DNA is continuously damaged by endogenous and exogenous factors such as oxidative stress or DNA alkylating agents. These damaged nucleobases are removed by DNA N-glycosylase and form apurinic/apyrimidinic sites (AP sites) as intermediates in the base excision repair (BER) pathway. AP sites are also representative DNA damages formed by spontaneous hydrolysis. The AP sites block DNA polymerase and a mismatch nucleobase is inserted opposite the AP sites by polymerization to cause acute toxicities and mutations. Thus, AP site specific compounds have attracted much attention for therapeutic and diagnostic purposes. In this study, we have developed nucleobase-polyamine conjugates as the AP site binding ligand by expecting that the nucleobase part would play a role in the specific recognition of the nucleobase opposite the AP site by the Watson-Crick base pair formation and that the polyamine part should contribute to the access of the ligand to the AP site by a non-specific interaction to the DNA phosphate backbone. The nucleobase conjugated with 3,3'-diaminodipropylamine (A-ligand, G-ligand, C-ligand, T-ligand and U-ligand) showed a specific stabilization of the duplex containing the AP site depending on the complementary combination with the nucleobase opposite the AP site; that is A-ligand to T, G-ligand to C, C-ligand to G, T- and U-ligand to A. The thermodynamic binding parameters clearly indicated that the specific stabilization is due to specific binding of the ligands to the complementary AP site. These results have suggested that the complementary base pairs of the Watson-Crick type are formed at the AP site.     

Synthesis and properties of an oligodeoxynucleotide modified with a pyrene derivative at the 5'-phosphate.

The synthesis of an oligonucleotide (ODN) modified with pyrene (pyr) on the 5'-phosphate is described. The ODN and pyrene are joined through a linker composed of four methylene groups. Modification of the oligonucleotide was effected via condensation of the 2-cyanoethyl N,N-diisopropylphosphoramidite of 4-(1-pyrenyl)butanol (pyr-m4OPAm, 2) with the 5'-OH of an ODN. This derivative is suitable for incorporation into automated solid-phase DNA synthesis and was attached to the 5' terminus of the DNA chain through a phosphodiester linkage. The properties of the 5'-(pyr-m4)d(T)15 (3) and the duplex it formed with d(A)15 were investigated by fluorescence and absorbance spectroscopy. The pyrene fluorescence in the modified duplex was quenched 96.3% relative to an identical concentration of free 4-(1-pyrenyl)butanol. The ultraviolet spectrum of the 5'-(pyr-m4)-d(T)15 and 5'-(pyr-m4)-d(T)15-d-(A)15 modified duplex, in the 320-360-nm region, was red-shifted 6 nm relative to the free 4-(1-pyrenyl)-butanol. The Tm values of the unmodified and modified duplexes at 0.1 M NaCl were 34.9 and 41.9 degrees C, respectively. The pyrene-induced stabilization corresponds to a free energy change (delta delta G degrees) of -2.6 kcal/mol.     

A study of side reactions occurring during synthesis of oligodeoxynucleotides containing O6-alkyldeoxyguanosine residues at preselected sites

As part of our studies on the molecular mechanisms of mutation by carcinogens we have synthesized 12 oligonucleotides (15-mers) containing an O6-alkylguanine residue at a preselected position for use as primers in the enzymatic synthesis of biologically active DNA. Ten of these oligonucleotides are derived from a minus strand sequence carrying the modified nucleotide in the third codon of gene G of bacteriophage phi X174 DNA. Two others are derived from plus strand sequences carrying the modification in the 12th codon of the human Ha-ras protooncogene. During this work several potentially serious side reactions, which could complicate interpretation of mutagenesis data, were observed. This paper describes a detailed study of these reactions. Since we were unable to avoid undesirable side products, we developed simple chromatographic methods for detecting and removing them.     

Synthesis and properties of oligodeoxynucleotides containing a C-2 polyamine-bearing deoxyguanosine residue as artificial ribonuclease.

Oligodeoxynucleotides containing a 2-fluoro-2'-deoxyinosine residue substituting normal 2'-deoxyguanosine residue were synthesized. Upon treating with ethanol solution of polyamine, the fluorine atom in the oligomers were readily substituted with the polyamine. The thermal stabilities of the duplexes consisted of the polyamine-bearing oligomers and their cDNAs as well as their RNA cleaving activity were investigated.     

Synthesis and properties of oligodeoxynucleotides containing the analogue 2'-deoxy-4'-thiothymidine.

The 2'-deoxythymidine analogue 2'-deoxy-4'-thiothymidine has been incorporated, using standard methodology, into a series of dodecadeoxynucleotides containing the EcoRV restriction endonuclease recognition site (GATATC). The stability of these oligodeoxynucleotides and their ability to act as substrates for the restriction endonuclease and associated methylase have been compared with a normal unmodified oligodeoxynucleotide. No problems were encountered in the synthesis despite the presence of a potentially oxidisable sulfur atom in the sugar ring. The analogue had very little effect on the melting temperature of the self-complementary oligoeoxynucleotides so synthesised and all had a CD spectrum compatible with a B-DNA structure. The oligodeoxynucleotide containing one analogue in each strand within the recognition site, adjacent to the bond to be cleaved (i.e. GAXATC, where X is 2'-deoxy-4'-thiothymidine), was neither a substrate for the endonuclease nor was recognized by the associated methylase. When still within the recognition hexanucleotide but two further residues removed from the site of cleavage (i.e. GATAXC), the oligodeoxynucleotide was a poor substrate for both the endonuclease and methylase. Binding of the oligodeoxynucleotide to the endonuclease was unaffected but the kcat value was only 0.03% of the value obtained for the parent oligodeoxynucleotide. These results show that the incorporation of 2'-deoxy-4'-thionucleosides into synthetic oligodeoxynucleotides may shed light on subtle interactions between proteins and their normal substrates and may also show why 2'-deoxy-4'-thiothymidine itself is so toxic in cell culture.     

Synthesis and characterization of viologen-modified oligodeoxynucleotides.

Several viologen-tagged oligodeoxynucleotides in the form of covalent linkage at the specific site of the phosphorous backbone were synthesized and characterized. Incorporation of viologen molecules was confirmed by the 31P-NMR, 20% PAGE analysis, enzymatic degradation, and uv, ESR spectra of the derived cation radical. The Tm values and CD spectra of the modified strands with their complementary strands indicate that the introduction of the viologen molecule via linker arm causes no significant perturbation in duplex structure.     

Synthesis and properties of oligodeoxynucleotides containing 5-carboxy-2'-deoxycytidines

5-Carboxy-2'-deoxycytidine (dC(COO-)) was synthesized as an anion-carrier to seek a new possibility of modified oligodeoxynucleotides capable of stabilization of duplexes and triplexes. The base pairing properties of this compound were evaluated by use of ab initio calculations. These calculations suggest that the Hoogsteen-type base pair of dC(COO-)-G is less stable than that of the canonical C+-G pair and the Watson-Crick-type base pair of dC(COO-)-G is slightly more stable than the natural G-C base pair. The modified cytosine base showed a basicity similar to that of cytosine (pKa 4.2). It turned out that oligodeoxynucleotides 13mer and 14mer incorporating dC(COO-) could form duplexes with the complementary DNA oligomer, which were more stable than the unmodified duplex. In contrast, it formed a relatively unstable triplex with the target ds DNA.     

Synthesis and properties of oligodeoxyribonucleotides containing abasic site.

Dodecanucleotide, d(CGCXCGCGTGCG), containing abasic site at desired position in the sequence was synthesized by solid-phase triester method. The introduced moiety (X) WAS N-(2'-deoxy-beta-D-erythropentofuranosyl) formamide.     

Efficiency of incision of an AP site within clustered DNA damage by the major human AP endonuclease.

A major DNA lesion induced by ionizing radiation and formed on removal of oxidized base lesions by various glycosylases is an apurinic/apyrimidinic site (AP site). The presence of an AP site within clustered DNA damage, induced following exposure to ionizing radiation or radiomimetic anticancer agents, may present a challenge to the repair machinery of the cell, if the major human AP endonuclease, HAP1, does not efficiently incise the AP site. In this study, specific oligonucleotide constructs containing an AP site located at several positions opposite to another damage [5,6-dihydrothymine (DHT), 8-oxoG, AP site, or various types of single strand breaks] on the complementary strand were used to determine the relative efficiency of the purified HAP1 protein in incising an AP site(s) from clustered DNA damage. A base damage (DHT and 8-oxoG) on the opposite strand has little or no influence on the rate of incision of an AP site by HAP1. In contrast, the presence of either a second AP site or various types of single strand breaks, when located one or three bases 3' to the base opposite to the AP site, has a strong inhibitory effect on the efficiency of incision of an AP site. The efficiency of binding of HAP1 to an AP site is reduced by approximately 1 order of magnitude if a single strand break (SSB) is located one or three bases 3' to the site opposite to the AP site on the complementary strand. If the AP site and either a SSB or a second AP site are located at any of the other positions relative to each other, a double strand break may result.     

Physicochemical properties of phosphorothioate oligodeoxynucleotides.

We have recently shown that phosphorothioate (PS) oligodeoxynucleotide (ODN) analogs, unlike their normal congeners, exhibit significant anti-HIV activity (Matsukura et al., (1987) Proc. Natl. Acad. Sci. USA 84, 7706-7710). We now report the syntheses, melting temperatures (Tm), and nuclease susceptibilities of a series of phosphorothioate ODN analogs. These include all-PS duplexes, duplexes with one normal chain and the other chain either all-PS, or end-capped with several PS groups at both 3' and 5' ends. The DNase susceptibilities of the S-ODNs are much less than the normal phosphodiesters, but by contrast duplexes of poly-rA with S-dT40 are much more susceptible to RNase H digestion. The Tm's for AT base pairs of S-ODNs are significantly depressed relative to normals, while GC base pairs show much less Tm depression. The Tm's of S-dT oligomers with poly-rA are reduced relative to the duplexes with normal dA oligomers. These results have significance for the biological properties of these analogs as anti-message inhibitors of gene expression, and provide a rational basis for the S-dC/G sequences as potential effective anti-AIDS agents.     

122 Kinetics of apurinic/apyrimidinic endonuclease 1 (APE1) on an authentic AP site or an AP site analog

Our genomic DNA is endlessly exposed to a wide variety of exogenous and endogenous DNA-damaging agents. One of the most abundant DNA lesions is an apurinic/apyrimidinic (AP) site, which in vivo, can form spontaneously or through various cellular pathways, including the repair activity of DNA glycosylase enzymes (Wilson & Barsky, 2001). Persistence of these AP sites is both highly mutagenic and cytotoxic to the cell (Loeb & Preston, 1986). AP endonuclease 1 (APE1), an Mg²⁺ dependent enzyme, is the major human endonuclease responsible for incising the DNA backbone at AP sites. Repair to canonical duplex DNA is then completed by DNA polymerase and DNA ligase. Recently, APE1, in conjunction with delivery of DNA-damaging agents, has become a target for chemotherapeutic research with the aim to inhibit APE1 activity (Fishel & Kelley, 2007). Therefore, an understanding of APE1 activity and its molecular mechanism is essential. In vitro, the authentic AP site is highly unstable and can undergo β-elimination, leading to a strand break (Strauss, Beard, Patterson & Wilson, 1997). Due to the fragility of the AP site, stable AP site analogs, such as the reduced AP site or tetrahydrofuran (THF) site, are typically used to study APE1 (Maher & Bloom, 2007; Strauss, Beard, Patterson & Wilson, 1997). In this work, we have performed the first comprehensive kinetic study of APE1 acting on the authentic AP site as well the reduced AP site and THF AP site analog. Transient-state kinetic experiments reveal that the strand incision chemistry step is fast, upwards of ～700?s^?1 for all substrates, making APE1 one of the fastest DNA repair enzymes. Steady-state kinetic experiments reveal for each substrate, a slow, post chemistry step limits the steady-state rate. The steady-state rate for APE1 acting on authentic AP and AP-Red substrates is highly dependent on Mg²⁺ concentration, while the steady-state rate for THF site was not dependent on Mg²⁺ concentration. This comprehensive kinetic analysis reveal differences and similarities in the way APE1 processes the authentic AP site compared to AP site analogs. Furthermore, these differences require consideration when choosing AP site analogs to study APE1.     

Synthesis and hybridization properties of oligodeoxynucleotides containing 3'-deoxy-3'-C-methyleneuridine

3'-Deoxy-3'-C-methyleneuridine nucleoside 1 has been incorporated into oligodeoxynucleotides. Relative to the unmodified references, oligomers containing nucleoside 1 displayed reduced binding affinities towards complementary DNA and RNA with a tendency towards RNA-selective hybridization.     

Synthesis and properties of bile acid phosphoramidites 5'-tethered to antisense oligodeoxynucleotides against HCV.

Recently, we synthesized antisense oligonucleotides (AS-ODNs) directed against the non-coding-region (NCR) and the adjacent core region of the hepatitis C virus (HCV) RNA. Backbone modifications like phosphorothioates, methyl- and benzylphosphonates were introduced three at each end of the sequence. For improvement of liver specific drug targeting and/or hepatocellular uptake efficient AS-ODNs were covalently conjugated to biomolecules such as cholesterol or bile acids. The use of base-labile alkylphosphonates afforded mild conditions for deprotection of bile acid conjugated AS-ODNs. Here, we describe a convenient synthesis of new cholic acid and taurocholic acid phosphoramidites. Derivatization to taurocholic acid was effected directly before phosphitylation reaction, which is the last step of the phosphoramidite synthesis. These building blocks were coupled to the 5'-position of AS-ODNs in the last step of solid-phase synthesis. After mild deprotection, purification and characterization the properties of these modified AS-ODNs like their lipophilicity or their ability to form stable duplices to DNA and RNA were investigated. Enhanced lipophilicity and formation of stable duplices and heteroduplices makes bile acid conjugated AS-ODNs interesting as antiviral antisense therapeutics against HCV.     

Synthesis of oligodeoxynucleotides incorporating 2-N-carbamoylguanine and evaluation of the hybridization properties

Previously, we reported 2-N-carbamoylguanine (cmG) as a guanine analog. We further studied the synthetic protocol and hybridization properties of oligodeoxynucleotides (ODNs) incorporating cmG. These ODNs were synthesized using the phosphoramidite of cmG without protection of the 6-O position. However, the isolated products were contaminated with deacylated products having guanine in place of cmG. The detailed analysis of the synthetic process suggested that the deacylation resulted from the reaction of the carbamoyl moiety with capping reagents. Protection of the 6-O position suppressed the side reaction. The thermal stability of the DNA duplexes incorporating cmG was analyzed. An analysis of T_m values revealed that the base discrimination ability of cmG was comparable to or higher than that of the canonical guanine depending on the flanking bases.     

Synthesis and nucleosome structure of DNA containing a UV photoproduct at a specific site.

A strategy was developed to assemble nucleosomes specifically damaged at only one site and one structural orientation. The most prevalent UV photoproduct, a cis-syn cyclobutane thymine dimer (cs CTD), was chemically synthesized and incorporated into a 30 base oligonucleotide harboring the glucocorticoid hormone response element. This oligonucleotide was assembled into a 165 base pair double stranded DNA molecule with nucleosome positioning elements on each side of the cs CTD-containing insert. Proton NMR verified that the synthetic photoproduct is the cis-syn stereoisomer of the CTD. Moreover, two different pyrimidine dimer-specific endonucleases cut approximately 90% of the dsDNA molecules. This cleavage is completely reversed by photoreactivation with E. coli UV photolyase, further demonstrating the correct stereochemistry of the photoproduct. Nucleosomes were reconstituted by histone octamer exchange from chicken erythocyte core particles, and contained a unique translational and rotational setting of the insert on the histone surface. Hydroxyl radical footprinting demonstrates that the minor groove at the cs CTD is positioned away from the histone surface about 5 bases from the nucleosome dyad. Competitive gel-shift analysis indicates there is a small increase in histone binding energy required for the damaged fragment (DeltaDeltaG approximately 0.15 kcal/mol), which does not prevent complete nucleosome loading under our conditions. Finally, folding of the synthetic DNA into nucleosomes dramatically inhibits cleavage at the cs CTD by T4 endonuclease V and photoreversal by UV photolyase. Thus, specifically damaged nucleosomes can be experimentally designed for in vitro DNA repair studies.